IDO1 Signaling through GCN2 in a Subpopulation of Gr-1+ Cells Shifts the IFNγ/IL6 Balance to Promote Neovascularization.

In addition to immunosuppression, it is generally accepted that myeloid-derived suppressor cells (MDSC) also support tumor angiogenesis. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) has been implicated in promoting neovascularization through its positioning as a key regulatory node between the inflammatory cytokines IFNγ and IL6. Here, we report that within the heterogeneous expanse of Gr-1+ MDSCs, both IDO1 expression and the ability to elicit neovascularization in vivo were associated with a minor subset of autofluorescent, CD11blo cells. IDO1 expression was further restricted to a discrete, CD11c and asialo-GM1 double-positive subpopulation of these cells, designated here as IDVCs (IDO1-dependent vascularizing cells), due to the dominant role that IDO1 activity in these cells was found to play in promoting neovascularization. Mechanistically, the induction of IDO1 in IDVCs provided a negative-feedback constraint on the antiangiogenic effect of host IFNγ by intrinsically signaling for the production of IL6 through general control nonderepressible 2 (GCN2)-mediated activation of the integrated stress response. These findings reveal fundamental molecular and cellular insights into how IDO1 interfaces with the inflammatory milieu to promote neovascularization.

Peptide vaccination directed against IDO1-expressing immune cells elicits CD8+ and CD4+ T-cell-mediated antitumor immunity and enhanced anti-PD1 responses.

BACKGROUND: The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which subverts T-cell immunity at multiple levels, is itself subject to inherent T-cell reactivity. This intriguing deviation from central tolerance has been interpreted as counterbalancing IDO1-mediated immunosuppression. Based on this hypothesis, clinical studies employing an IDO1 peptide-based vaccine approach for cancer treatment have been initiated, but there remains a pressing need to further investigate the immunological ramifications of stimulating the anti-IDO1 T-cell response in this manner. METHODS: CT26 colon carcinoma tumors were evaluated for expression of IDO1 protein by western blot analysis, immunofluorescence microscopy and flow cytometry. Mouse IDO1-derived peptides, predicted to bind either major histocompatibility complex (MHC) class I or II of the H2d BALB/c strain, were emulsified in 50% Montanide for prophylactic or therapeutic vaccine treatment of CT26 tumor-bearing mice initiated either 7 days prior to or following tumor cell injection, respectively. In some therapeutic treatment experiments, administration of programmed cell death protein 1-binding antibody (anti-PD1 antibody) or epacadostat was concurrently initiated. Tumor size was determined by caliper measurements and comparative tumor growth suppression was assessed by longitudinal analyses of tumor growth data. For adoptive transfer, T cells from complete responder animals were isolated using paramagnetic beads and fluorescence-activated cell sorting. RESULTS: This study identifies mouse MHC class I-directed and II-directed, IDO1-derived peptides capable of eliciting antitumor responses, despite finding IDO1 expressed exclusively in tumor-infiltrating immune cells. Treatment of established tumors with anti-PD1 antibody and class I-directed but not class II-directed IDO1 peptide vaccines produced an enhanced antitumor response. Likewise, class I-directed and II-directed IDO1 peptides elicited an enhanced combinatorial response, suggesting distinct mechanisms of action. Consistent with this interpretation, adoptive transfer of isolated CD8+ T cells from class I and CD4+ T cells from class II peptide-vaccinated responder mice delayed tumor growth. The class II-directed response was completely IDO1-dependent while the class I-directed response included an IDO1-independent component consistent with antigen spread. CONCLUSIONS: The in vivo antitumor effects demonstrated with IDO1-based vaccines via targeting of the tumor microenvironment highlight the utility of mouse models for further exploration and refinement of this novel vaccine-based approach to IDO1-directed cancer therapy and its potential to improve patient response rates to anti-PD1 therapy.

Host IDO2 Gene Status Influences Tumor Progression and Radiotherapy Response in KRAS-Driven Sporadic Pancreatic Cancers.

PURPOSE: Heritable genetic variations can affect the inflammatory tumor microenvironment, which can ultimately affect cancer susceptibility and clinical outcomes. Recent evidence indicates that IDO2, a positive modifier in inflammatory disease models, is frequently upregulated in pancreatic ductal adenocarcinoma (PDAC). A unique feature of IDO2 in humans is the high prevalence of two inactivating single-nucleotide polymorphisms (SNP), which affords the opportunity to carry out loss-of-function studies directly in humans. In this study, we sought to address whether genetic loss of IDO2 may influence PDAC development and responsiveness to treatment.Experimental Design: Transgenic Ido2 +/+ and Ido2 -/- mice in which oncogenic KRAS is activated in pancreatic epithelial cells were evaluated for PDAC. Two patient data sets (N = 200) were evaluated for the two IDO2-inactivating SNPs together with histologic, RNA expression, and clinical survival data. RESULTS: PDAC development was notably decreased in the Ido2 -/- mice (30% vs. 10%, P < 0.05), with a female predominance similar to the association observed for one of the human SNPs. In patients, the biallelic occurrence of either of the two IDO2-inactivating SNPs was significantly associated with markedly improved disease-free survival in response to adjuvant radiotherapy (P < 0.01), a treatment modality that has been highly debated due to its variable efficacy. CONCLUSIONS: The results of this study provide genetic support for IDO2 as a contributing factor in PDAC development and argue that IDO2 genotype analysis has the immediate potential to influence the PDAC care decision-making process through stratification of those patients who stand to benefit from adjuvant radiotherapy.

IDO1 is an Integral Mediator of Inflammatory Neovascularization.

The immune tolerogenic effects of IDO1 (indoleamine 2,3-dioxygenase 1) have been well documented and genetic studies in mice have clearly established the significance of IDO1 in tumor promotion. Dichotomously, the primary inducer of IDO1, the inflammatory cytokine IFNγ (interferon-γ), is a key mediator of immune-based tumor suppression. One means by which IFNγ can exert an anti-cancer effect is by decreasing tumor neovascularization. We speculated that IDO1 might contribute to cancer promotion by countering this anti-neovascular effect of IFNγ, possibly through IDO1-potentiated elevation of the pro-tumorigenic inflammatory cytokine IL6 (interleukin-6). In this study, we investigated how genetic loss of IDO1 affects neovascularization in mouse models of oxygen-induced retinopathy and lung metastasis. Neovascularization in both models was significantly reduced in mice lacking IDO1, was similarly reduced with loss of IL6, and was restored in both cases by concomitant loss of IFNγ. Likewise, the lack of IDO1 or IL6 resulted in reduced metastatic tumor burden and increased survival, which the concomitant loss of IFNγ abrogated. This insight into IDO1's involvement in pro-tumorigenic inflammatory neovascularization may have important ramifications for IDO1 inhibitor development, not only in cancer where clinical trials are currently ongoing, but in other disease indications associated with neovascularization as well.

IDO is a nodal pathogenic driver of lung cancer and metastasis development.

UNLABELLED: Indoleamine 2,3-dioxygenase (IDO) enzyme inhibitors have entered clinical trials for cancer treatment based on preclinical studies, indicating that they can defeat immune escape and broadly enhance other therapeutic modalities. However, clear genetic evidence of the impact of IDO on tumorigenesis in physiologic models of primary or metastatic disease is lacking. Investigating the impact of Ido1 gene disruption in mouse models of oncogenic KRAS-induced lung carcinoma and breast carcinoma-derived pulmonary metastasis, we have found that IDO deficiency resulted in reduced lung tumor burden and improved survival in both models. Micro-computed tomographic (CT) imaging further revealed that the density of the underlying pulmonary blood vessels was significantly reduced in Ido1-nullizygous mice. During lung tumor and metastasis outgrowth, interleukin (IL)-6 induction was greatly attenuated in conjunction with the loss of IDO. Biologically, this resulted in a consequential impairment of protumorigenic myeloid-derived suppressor cells (MDSC), as restoration of IL-6 recovered both MDSC suppressor function and metastasis susceptibility in Ido1-nullizygous mice. Together, our findings define IDO as a prototypical integrative modifier that bridges inflammation, vascularization, and immune escape to license primary and metastatic tumor outgrowth. SIGNIFICANCE: This study provides preclinical, genetic proof-of-concept that the immunoregulatory enzyme IDO contributes to autochthonous carcinoma progression and to the creation of a metastatic niche. IDO deficiency in vivo negatively impacted both vascularization and IL-6­dependent, MDSC-driven immune escape, establishing IDO as an overarching factor directing the establishment of a protumorigenic environment.

Non-hematopoietic expression of IDO is integrally required for inflammatory tumor promotion.

Indoleamine 2,3-dioxygenase (IDO) is generally considered to be immunosuppressive but recent findings suggest this characterization oversimplifies its role in disease pathogenesis. Recently, we showed that IDO is essential for tumor outgrowth in the classical two-stage model of inflammatory skin carcinogenesis. Here, we report that IDO loss did not exacerbate classical inflammatory responses. Rather, IDO induction could be elicited by environmental signals and tumor promoters as an integral component of the inflammatory tissue microenvironment even in the absence of cancer. IDO loss had limited impact on tumor outgrowth in carcinogenesis models that lacked an explicit inflammatory tumor promoter. In the context of inflammatory carcinogenesis where IDO was critical to tumor development, the most important source of IDO was radiation-resistant non-hematopoietic cells, consistent with evidence that loss of the IDO regulatory tumor suppressor gene Bin1 in transformed skin cells facilitates IDO-mediated immune escape by a cell autonomous mechanism. Taken together, our results identify IDO as an integral component of 'cancer-associated' inflammation that tilts the immune system toward tumor support. More generally, they promote the concept that mediators of immune escape and cancer-associated inflammation may be genetically synonymous.

Bin3 deletion causes cataracts and increased susceptibility to lymphoma during aging.

Bin3 encodes an evolutionarily conserved and ubiquitously expressed member of the BAR superfamily of curved membrane and GTPase-binding proteins, which includes the BAR, PCH/F-BAR, and I-BAR adapter proteins implicated in signal transduction and vesicular trafficking. In humans, Bin3 maps to chromosome 8p21.3, a region widely implicated in cancer suppression that is often deleted in non-Hodgkin's lymphomas and various epithelial tumors. Yeast studies have suggested roles for this gene in filamentous actin (F-actin) organization and cell division but its physiologic functions in mammals have not been investigated. Here we report that homozygous inactivation of Bin3 in the mouse causes cataracts and an increased susceptibility to lymphomas during aging. The cataract phenotype was marked by multiple morphologic defects in lens fibers, including the development of vacuoles in cortical fibers and a near total loss of F-actin in lens fiber cells but not epithelial cells. Through 1 year of age, no other phenotypes were apparent; however, by 18 months of age, Bin3(-/-) mice exhibited a significantly increased incidence of lymphoma. Bin3 loss did not affect normal cell proliferation, F-actin organization, or susceptibility to oncogenic transformation. In contrast, it increased the proliferation and invasive motility of cells transformed by SV40 large T antigen plus activated ras. Our findings establish functions for Bin3 in lens development and cancer suppression during aging. Further, they define Bin3 as a candidate for an unidentified tumor suppressor that exists at the human chromosome 8p21.3 locus.

Bin1 ablation in mammary gland delays tissue remodeling and drives cancer progression.

Genes that modify oncogenesis may influence dormancy versus progression in cancer, thereby affecting clinical outcomes. The Bin1 gene encodes a nucleocytosolic adapter protein that interacts with and suppresses the cell transforming activity of Myc. Bin1 is often attenuated in breast cancer but its ability to negatively modify oncogenesis or progression in this context has not been gauged directly. In this study, we investigated the effects of mammary gland-specific deletion of Bin1 on initiation and progression of breast cancer in mice. Bin1 loss delayed the outgrowth and involution of the glandular ductal network during pregnancy but had no effect on tumor susceptibility. In contrast, in mice where tumors were initiated by the ras-activating carcinogen 7,12-dimethylbenz(a)anthracene, Bin1 loss strongly accentuated the formation of poorly differentiated tumors characterized by increased proliferation, survival, and motility. This effect was specific as Bin1 loss did not accentuate progression of tumors initiated by an overexpressed mouse mammary tumor virus-c-myc transgene, which on its own produced poorly differentiated and aggressive tumors. These findings suggest that Bin1 loss cooperates with ras activation to drive progression, establishing a role for Bin1 as a negative modifier of oncogenicity and progression in breast cancer.

Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy.

Immune escape is a crucial feature of cancer progression about which little is known. Elevation of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) in tumor cells can facilitate immune escape. Not known is how IDO becomes elevated or whether IDO inhibitors will be useful for cancer treatment. Here we show that IDO is under genetic control of Bin1, which is attenuated in many human malignancies. Mouse knockout studies indicate that Bin1 loss elevates the STAT1- and NF-kappaB-dependent expression of IDO, driving escape of oncogenically transformed cells from T cell-dependent antitumor immunity. In MMTV-Neu mice, an established breast cancer model, we show that small-molecule inhibitors of IDO cooperate with cytotoxic agents to elicit regression of established tumors refractory to single-agent therapy. Our findings suggest that Bin1 loss promotes immune escape in cancer by deregulating IDO and that IDO inhibitors may improve responses to cancer chemotherapy.

Targeted deletion of the suppressor gene bin1/amphiphysin2 accentuates the neoplastic character of transformed mouse fibroblasts.

The Bin1/Amphiphysin2 gene encodes several alternately spliced BAR adapter proteins that have been implicated in membrane-associated and nuclear processes. Bin1 expression is often attenuated during tumor progression and Bin1 splice isoforms that localize to the nucleus display tumor suppressor properties. While these properties may reflect the ability of these isoforms to interact with and suppress the cell transforming activity of c-Myc, the effects of Bin1 deletion on the oncogenicity of c-myc or other transforming genes has not been gauged directly. Here we report that targeted deletion of Bin1 enhances the neoplastic character of primary murine embryo fibroblasts (MEFs) cotransformed by c-myc and mutant grasg. Specifically, Bin1 loss accentuated the spindle morphology of transformed cells, increased anchorage-independent proliferation, and promoted tumor formation in syngeneic hosts. These effects were specific as they were not recapitulated in cells transformed by viral oncoproteins and mutant ras. Although some Bin1 splice isoforms associate with endocytotic complexes the effects of Bin1 loss were not correlated with a generalized defect in receptor-mediated endocytosis. However, Bin1 loss increased sensitivity to paclitaxel, a drug that can affect endocytotic trafficking by disrupting microtubule dynamics. In E1A?transformed MEFs, Bin1 loss reduced the susceptibility to apoptosis triggered by tumor necrosis factor-alpha, an effect that was associated with precocious nuclear trafficking of NF-kappaB. These findings offer a novel line of support for the hypothesized role of Bin1 in limiting malignant growth, possibly as a negative modifier or anti-progression gene.